COMPOSITIONS INCLUDING A VACANCY-ENGINEERED (VE)-ZnO  NANOCOMPOSITE, METHODS OF MAKING THE COMPOSITIONS AND METHODS  OF USING THE COMPOSITIONS

ABSTRACT

Embodiments of the present disclosure, in one aspect, relate to compositions including a vacancy-engineered (VE)-ZnO nanocomposite, methods of making a composition, methods of using a composition, and the like.

CROSS-REFERENCE TO RELATED APPLICATION

This application is related to, and derives from, United StatesProvisional patent application Ser. No. 62/119,494, filed 23 Feb. 2015and titled “Compositions Including a Vacancy-Engineered (VE)-ZNONanocomposite, Methods of Making a Composition, Methods of Using aComposition.”

BACKGROUND

The globalization of business, travel and communication brings increasedattention to worldwide exchanges between communities and countries,including the potential globalization of the bacterial and pathogenicecosystem. Bactericides and fungicides have been developed to controldiseases in man, animals, and plants, and must evolve to remaineffective as more and more antibiotic, pesticide, and insecticideresistant bacteria and fungi appear around the globe.

Bacterial resistance to antimicrobial agents has also emerged,throughout the world, as one of the major threats to both man and theagrarian lifestyle. Resistance to antibacterial and antifungal agentshas emerged as an agricultural issue that requires attention andimprovements in the treatment materials in use today.

For example, focusing on plants, there are over 300,000 diseases thatafflict plants worldwide, resulting in billions of dollars of annualcrop losses. The antibacterial/antifungal formulations in existencetoday could be improved and made more effective.

SUMMARY

Embodiments of the present disclosure, in one aspect, relate tocompositions including a vacancy-engineered (VE)-ZnO nanocomposite,methods of making the composition, methods of using the composition, andthe like.

In an embodiment, a composition, among others, includes: avacancy-engineered (VE)-ZnO nanocomposite including a plurality ofinterconnected VE-ZnO nanoparticles, wherein the plurality of VE-ZnOnanoparticles has a plurality of surface defects associated with anoxygen vacancy, wherein at least either: (1) the plurality of VE-ZnOnanoparticles each has a diameter of other than about 3 to 8 nm; orwherein (2) the plurality of VE-ZnO nanoparticles each does not includesa coating of a surface capping agent having one or more Zn ion chelatingfunctional groups.

In an embodiment, a method, among others, includes: disposing acomposition on a surface, wherein the composition has avacancy-engineered (VE)-ZnO nanocomposite including a plurality ofinterconnected VE-ZnO nanoparticles, wherein the plurality ofinterconnected VE-ZnO nanoparticles has a plurality of surface defectsassociated with an oxygen vacancy, wherein at least either: (1) theplurality of VE-ZnO nanoparticles each does not have a diameter of about3 to 8 nm; or wherein (2) the plurality of VE-ZnO nanoparticles eachdoes not include a coating of a surface capping agent having one or moreZn ion chelating functional groups; and killing a substantial portion ofa microorganism or inhibiting or substantially inhibiting the growth ofthe microorganisms on the surface of a structure or that come intocontact with the surface of the structure.

In an embodiment, a method, among others, includes: mixing a watersoluble zinc source, a surface capping agent, and an oxidizing agent,wherein the surface capping agent has both a carboxyl group and hydroxylgroup; and forming a vacancy-engineered (VE)-ZnO nanocomposite includinga plurality of interconnected VE-ZnO nanoparticles, wherein theplurality of VE-ZnO nanoparticles has surface defects associated with anoxygen vacancy, wherein at least either: (1) the plurality of VE-ZnOnanoparticles has a diameter of other than about 1 to 10 nm; or wherein(2) the plurality of VE-ZnO nanoparticles does not include a coatingformed from the surface capping agent.

Other compositions, methods, features, and advantages will be, orbecome, apparent to one with skill in the art upon examination of thefollowing drawings and detailed description.

The embodiments contemplate that compositions, methods, features andadvantages may include compositions that may be defined with a limitednumber of limitations, or negative limitations, as presented anddescribed above. It is intended that all such additional structures,compositions, methods, features, and advantages be included within thisdescription, be within the scope of the present disclosure, and beprotected by the accompanying claims. A particular composition inaccordance with the disclosure with such fewer limitations includes acomposition that, among other compositions, includes: avacancy-engineered (VE)-ZnO nanocomposite including a plurality ofinterconnected VE-ZnO nanoparticles, wherein the plurality of VE-ZnOnanoparticles has a plurality of surface defects associated with anoxygen vacancy, with the particle size and surface capping agentlimitations as described above. The disclosure also contemplates relatedmethods for use of or preparation of the composition.

The disclosure contemplates that the VE-ZnO nanoparticle size rangeother than about 3 to 8 nm or other than about 1 to 10 nm may beencompassed by a particle range of greater than about 10 nm to about 100nm, or alternatively greater than about 10 nm to about 200 nm or furtheralternatively greater than about 10 nm to about 500 nm. Alternativelyconsidered is a range from about 25 to about 500 nm or alternativelyfrom about 50 to about 500 nm. Upper size ranges of up to about 1 micronare considered. By excluding the size range from 1 to 10 nm and 3 to 8nm it is intended to illustrate that efficacy of a composition inaccordance with the disclosure is not necessarily limited to a smallsize range which has particularly desirable characteristics.

The disclosure also contemplates as operative VE-ZnO nanoparticle sizessmaller than about 1 nm or smaller than about 0.5 nm, either of whichmay serve as an upper limit in a range having a lower limit bounded byabout 0.1 nm.

By excluding the coating formed of the surface capping agent thedisclosure is intended to include as viable compositions less complexcompositions that include zinc oxide materials that include oxygenmaterials derived from peroxide materials and hydroxide materials, butabsent a layer formed of a surface capping agent.

BRIEF DESCRIPTION OF THE DRAWINGS

Many aspects of this disclosure can be better understood with referenceto the following drawings. The components in the drawings are notnecessarily to scale, emphasis instead being placed upon clearlyillustrating the principles of the present disclosure. Moreover, in thedrawings, like reference numerals designate corresponding partsthroughout the several views.

FIG. 1 illustrates: (a) A representative HRTEM image of Zinkicide™ SG4showing plate-like faceted structure in the sub-micron size range. (b)High-magnification image of coated ZnO material shows appearance of bothpolycrystalline and amorphous regions within a plate structure. FieldEmission Scanning Electron Microscopy (FE-SEM) image of the material areshown in image (c) and (d).

FIG. 2 illustrates: (a) A representative low-magnification HRTEM imageof Zinkicide™ SG6 material showing gel-like network of inter-connectingultra-small size (<5 nm) crystalline sol particle clusters. (b)High-magnification image of Zinkicide™ SG6 material. Inset showscrystalline lattice fringe of one of Zinkicide™ SG6 sol particles. Note:one nm is a billionth of a meter. Field Emission Scanning ElectronMicroscopy (FE-SEM) images of the material are shown in image (c) and(d).

FIG. 3 illustrates: (a) A representative HRTEM image of Nordox 30/30 WGmaterial showing polydispersed structure in the size ranging from nanoto micron size. (b) High-magnification image of Nordox material showsappearance of highly crystalline structure (see inset; HRTEM-SAEDpattern showing bright spots confirming crystallinity). Field EmissionScanning Electron Microscopy (FE-SEM) images of the material are shownin image (c) and (d).

FIGS. 4A through 4E illustrate comparative phytotoxicity results ofvarious coatings upon vinca plant.

FIG. 5 illustrates the growth inhibition with Alamar blue Assay of E.coli against VE-ZnO, coated ZnO, Nordox, and Kocide 3000.

FIG. 6 illustrates E. coli growth curves in presence of Zinkicide™against VE-ZnO, coated ZnO, Nordox, and Kocide 3000.

FIG. 7 illustrates E. coli viability in presence of Zinkicide™materials.

FIG. 8 illustrates direct evidence of reactive oxygen species (ROS)generation by the coated VE-ZnO material.

FIGS. 9A and 9B illustrate HRTEM-EDX spectra of surface coated VE-ZnOand ZnO.

FIGS. 10A and 10B illustrate x-ray photoelectron spectroscopy (XPS)results of surface coated VE-ZnO and ZnO.

FIG. 11 illustrates a schematic representation of VE-ZnO (“Zinkicide”)nanoparticle composite (nanocomposite).

FIG. 12 illustrates rainfastness data of VE-ZnO nanoparticle composites.

FIG. 13 illustrates tabular data for germination of VE-ZnO treated snowpea seeds.

FIG. 14A, 14B and 14C illustrate experimental design and experimentaldata for tomato plants treated with VE-ZnO nanoparticle composite.

FIG. 15 illustrate UV-visible absorbance spectra for VE-ZnO nanoparticlecomposite, where the intersection points with the vertical axis from lowto high absorbance correspond with sodium salicylate, ZnO, Zinkicide SG4and Zinkicide SG6.

FIG. 16A and 16B illustrate fluorescence emission spectra for VE-ZnOnanoparticle composites SG4 and SG6.

FIG. 17A and 17B illustrate FT-IR spectra of surface coated ZnO, surfacecoated VE-ZnO and the surface coating agent. FTIR results show that thesurface coating agent is present in both ZnO and VE-ZnO materials. InFIG. 17A, the curve that corresponds with the peak at 1600 cm⁻¹corresponds with the surface coating agent. The curve that correspondswith the peak at 1350 cm⁻¹ corresponds with surface coated ZnO and theremaining curve which does not include a deep peak corresponds withsurface coated VE-ZnO. In FIG. 17B, the curve that corresponds with thepeaek at 3500 cm⁻¹ cororesponds with the surface coated VE-ZnO, thecurve that corresponds with thet peak at 2000 cm⁻¹ corrresponds withsurface coating agent and the remaining curve corresponds with thesurface coated VE-ZnO.

FIG. 18 illustrates an XRD of surface coated VE-ZnO. XRD patternrevealing 200 (strong), 220 and 311 reflection peaks VE-ZnO at 2 ⊖ valueof ˜36°, 54° and 64° were observed. These peaks are characteristic toZnO material with oxygen vacancy. The appearance of XRD peak at 2⊖ valueof ˜17° has not been assigned yet (possibly originating from the surfacecoating agent).

FIG. 19A and 19B illustrate Raman spectra of (a) surface coated VE-ZnOand (b) surface coated ZnO materials. Appearance of strong ˜840 cm⁻¹Raman peak is characteristic to VE-ZnO O—O stretching vibration ofperoxide (an active ROS). No such peak is present in surface coated ZnOmaterial.

FIG. 20A and 20B illustrate DLS particle size distribution of (a)surface coated ZnO and (b) surface coated VE-ZnO materials. Narrowparticle size distribution of VE-ZnO material is indicative of smallerand uniform-size cluster formation.

DETAILED DESCRIPTION

Before the present disclosure is described in greater detail, it is tobe understood that this disclosure is not limited to particularembodiments described, and as such may, of course, vary. It is also tobe understood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present disclosure will be limited onlyby the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present disclosure, the preferredmethods and materials are now described.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present disclosure is not entitled to antedate suchpublication by virtue of prior disclosure. Further, the dates ofpublication provided could be different from the actual publicationdates that may need to be independently confirmed.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features that may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentdisclosure. Any recited method can be carried out in the order of eventsrecited or in any other order that is logically possible.

Embodiments of the present disclosure will employ, unless otherwiseindicated, techniques of chemistry, polymer chemistry, biology, and thelike, which are within the skill of the art. Such techniques areexplained fully in the literature.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how toperform the methods and use the compositions and compounds disclosed andclaimed herein. Efforts have been made to ensure accuracy with respectto numbers (e.g., amounts, temperature, etc.), but some errors anddeviations should be accounted for. Unless indicated otherwise, partsare parts by weight, temperature is in ° C., and pressure is inatmospheres. Standard temperature and pressure are defined as 25° C. and1 atmosphere.

Before the embodiments of the present disclosure are described indetail, it is to be understood that, unless otherwise indicated, thepresent disclosure is not limited to particular materials, reagents,reaction materials, manufacturing processes, or the like, as such canvary. It is also to be understood that the terminology used herein isfor purposes of describing particular embodiments only, and is notintended to be limiting. It is also possible in the present disclosurethat steps can be executed in different sequence where this is logicallypossible.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to “a support” includes a plurality of supports. In thisspecification and in the claims that follow, reference will be made to anumber of terms that shall be defined to have the following meaningsunless a contrary intention is apparent.

Definitions (Which are Not Necessarily Limited to the PresentDisclosure):

The term “antimicrobial characteristic” refers to the ability to killand/or inhibit the growth of microorganisms. A substance having anantimicrobial characteristic may be harmful to microorganisms (e.g.,bacteria, fungi, protozoans, algae, and the like). A substance having anantimicrobial characteristic can kill the microorganism and/or preventor substantially prevent or inhibit the growth or reproduction of themicroorganism.

The term “antibacterial characteristic” refers to the ability to killand/or inhibit the growth of bacteria. A substance having anantibacterial characteristic may be harmful to bacteria. A substancehaving an antibacterial characteristic can kill the bacteria and/orprevent or substantially prevent or inhibit the replication orreproduction of the bacteria.

“Gel matrix” or “Nanogel matrix” refers to amorphous gel like substancethat is formed by the interconnection of vacancy engineered crystallinezinc oxide nanoparticles (e.g., about 3 to 8 nm) to one another. In anembodiment, the amorphous gel matrix has no ordered (e.g., defined)structure. In an embodiment, the vacancy engineered zinc oxidenanoparticles are interconnected covalently (e.g., through —Zn—O—Zn—bonds), physically associated via Van der Waal forces, and/or throughionic interactions.

“Uniform plant surface coverage” refers to a uniform and complete (e.g.,about 100%) wet surface due to spray application of embodiments of thepresent disclosure. In other words, spray application causes embodimentsof the present disclosure to spread throughout the plant surface.

“Substantial uniform plant surface coverage” refers to about 70% ormore, about 80% or more, about 90% or more, or more uniform plantsurface coverage. “Substantially covering” refers to covering about 70%or more, about 80% or more, about 90% or more, or more, of the leavesand branches of a plant.

“Plant” refers to trees, plants, shrubs, flowers, and the like as wellas portions of the plant such as twigs, leaves, stems, branches, fruit,flowers, and the like. In a particular embodiment, the term plantincludes a fruit tree such as a citrus tree (e.g., orange tree, lemontree, lime tree, and the like).

As used herein, “treat,” “treatment,” “treating,” and the like refer toacting upon a disease or condition with a composition of the presentdisclosure to affect the disease or condition by improving or alteringit. In addition, “treatment” includes completely or partially preventing(e.g., about 70% or more, about 80% or more, about 90% or more, about95% or more, or about 99% or more) a plant form acquiring a disease orcondition. The phrase “prevent” can be used instead of treatment forthis meaning. “Treatment,” as used herein, covers one or more treatmentsof a disease in a plant, and includes: (a) reducing the risk ofoccurrence of the disease in a plant predisposed to the disease but notyet diagnosed as infected with the disease (b) impeding the developmentof the disease, and/or (c) relieving the disease, e.g., causingregression of the disease and/or relieving one or more disease symptoms.

As used herein, the terms “application,” “apply,” and the like, withinthe context of the terms “treat,” “treatment,” “treating” or the like,refers to the placement or introduction of a composition of thedisclosure onto or into a “plant” in accordance with the disclosure sothat the composition in accordance with the disclosure may “treat” aplant disease in accordance with the disclosure. The DetailedDescription of the Embodiments specifically teach: (1) a foliar“application” through use of a spray method or a drench method withrespect to a “plant” leaf; or (2) a root “application” through the spraymethod or the drench method with respect to a growth medium. Within thisdisclosure an “application” is intended to be broadly interpreted toinclude any extrinsic method or activity that provides for, or resultsin, introduction of a composition in accordance with the disclosure ontoor into a “plant” in accordance with the disclosure. Such methods oractivities may include, but are not necessarily limited to spraymethods, drench methods and hypodermic or other injection methods.

The terms “bacteria” or “bacterium” include, but are not limited to,Gram positive and Gram negative bacteria. Bacteria can include, but arenot limited to, Abiotrophia, Achromobacter, Acidaminococcus, Acidovorax,Acinetobacter, Actinobacillus, Actinobaculum, Actinomadura, Actinomyces,Aerococcus, Aeromonas, Afipia, Agrobacterium, Alcaligenes, Alloiococcus,Alteromonas, Amycolata, Amycolatopsis, Anaerobospirillum, Anabaenaaffinis and other cyanobacteria (including the Anabaena, Anabaenopsis,Aphanizomenon, Camesiphon, Cylindrospermopsis, Gloeobacter Hapalosiphon,Lyngbya, Microcystis, Nodularia, Nostoc, Phormidium, Planktothrix,Pseudoanabaena, Schizothrix, Spirulina, Trichodesmium, and Umezakiagenera) Anaerorhabdus, Arachnia, Arcanobacterium, Arcobacter,Arthrobacter, Atopobium, Aureobacterium, Bacteroides, Balneatrix,Bartonella, Bergeyella, Bifidobacterium, Bilophila Branhamella,Borrelia, Bordetella, Brachyspira, Brevibacillus, Brevibacterium,Brevundimonas, Brucella, Burkholderia, Buttiauxella, Butyrivibrio,Calymmatobacterium, Campylobacter, Capnocytophaga, Cardiobacterium,Catonella, Cedecea, Cellulomonas, Centipeda, Chlamydia, Chlamydophila,Chromobacterium, Chyseobacterium, Chryseomonas, Citrobacter,Clostridium, Collinsella, Comamonas, Corynebacterium, Coxiella,Cryptobacterium, Delftia, Dermabacter, Dermatophilus, Desulfomonas,Desulfovibrio, Dialister, Dichelobacter, Dolosicoccus, Dolosigranulum,Edwardsiella, Eggerthella, Ehrlichia, Eikenella, Empedobacter,Enterobacter, Enterococcus, Erwinia, Erysipelothrix, Escherichia,Eubacterium, Ewingella, Exiguobacterium, Facklamia, Filifactor,Flavimonas, Flavobacterium, Francisella, Fusobacterium, Gardnerella,Gemella, Globicatella, Gordona, Haemophilus, Hafnia, Helicobacter,Helococcus, Holdemania Ignavigranum, Johnsonella, Kingella, Klebsiella,Kocuria, Koserella, Kurthia, Kytococcus, Lactobacillus, Lactococcus,Lautropia, Leclercia, Legionella, Leminorella, Leptospira, Leptotrichia,Leuconostoc, Listeria, Listonella, Megasphaera, Methylobacterium,Microbacterium, Micrococcus, Mitsuokella, Mobiluncus, Moellerella,Moraxella, Morganella, Mycobacterium, Mycoplasma, Myroides, Neisseria,Nocardia, Nocardiopsis, Ochrobactrum, Oeskovia, Oligella, Orientia,Paenibacillus, Pantoea, Parachlamydia, Pasteurella, Pediococcus,Peptococcus, Peptostreptococcus, Photobacterium, Photorhabdus,Phytoplasma, Plesiomonas, Porphyrimonas, Prevotella, Propionibacterium,Proteus, Providencia, Pseudomonas, Pseudonocardia, Pseudoramibacter,Psychrobacter, Rahnella, Ralstonia, Rhodococcus, Rickettsia RochalimaeaRoseomonas, Rothia, Ruminococcus, Salmonella, Selenomonas, Serpulina,Serratia, Shewenella, Shigella, Simkania, Slackia, Sphingobacterium,Sphingomonas, Spirillum, Spiroplasma, Staphylococcus, Stenotrophomonas,Stomatococcus, Streptobacillus, Streptococcus, Streptomyces,Succinivibrio, Sutterella, Suttonella, Tatumella, Tissierella,Trabulsiella, Treponema, Tropheryma, Tsakamurella, Turicella,Ureaplasma, Vagococcus, Veillonella, Vibrio, Weeksella, Wolinella,Xanthomonas, Xenorhabdus, Yersinia, and Yokenella. Other examples ofbacterium include Mycobacterium tuberculosis, M bovis, M typhimurium, Mbovis strain BCG, BCG substrains, M avium, M intracellulare, Mafricanum, M kansasii, M marinum, M ulcerans, M avium subspeciesparatuberculosis, Staphylococcus aureus, Staphylococcus epidermidis,Staphylococcus equi, Streptococcus pyogenes, Streptococcus agalactiae,Listeria monocytogenes, Listeria ivanovii, Bacillus anthracis, B.subtilis, Nocardia asteroides, and other Nocardia species, Streptococcusviridans group, Peptococcus species, Peptostreptococcus species,Actinomyces israelii and other Actinomyces species, andPropionibacterium acnes, Clostridium tetani, Clostridium botulinum,other Clostridium species, Pseudomonas aeruginosa, other Pseudomonasspecies, Campylobacter species, Vibrio cholera, Ehrlichia species,Actinobacillus pleuropneumonias, Pasteurella haemolytica, Pasteurellamultocida, other Pasteurella species, Legionella pneumophila, otherLegionella species, Salmonella typhi, other Salmonella species, Shigellaspecies Brucella abortus, other Brucella species, Chlamydi trachomatis,Chlamydia psittaci, Coxiella burnetti, Escherichia coli, Neiserriameningitidis, Neiserria gonorrhea, Haemophilus influenzae, Haemophilusducreyi, other Hemophilus species, Yersinia pestis, Yersiniaenterolitica, other Yersinia species, Escherichia coli, E. hirae andother Escherichia species, as well as other Enterobacteria, Brucellaabortus and other Brucella species, Burkholderia cepacia, Burkholderiapseudomallei, Francisella tularensis, Bacteroides fragilis,Fudobascterium nucleatum, Provetella species, and Cowdria ruminantium,or any strain or variant thereof. The Gram-positive bacteria mayinclude, but is not limited to, Gram positive Cocci (e.g.,Streptococcus, Staphylococcus, and Enterococcus). The Gram-negativebacteria may include, but is not limited to, Gram negative rods (e.g.,Bacteroidaceae, Enterobacteriaceae, Vibrionaceae, Pasteurellae andPseudomonadaceae). In an embodiment, the bacteria can include Mycoplasmapneumoniae.

The term “protozoan” as used herein includes, without limitationsflagellates (e.g., Giardia lamblia), amoeboids (e.g., Entamoebahistolitica), and sporozoans (e.g., Plasmodium knowlesi) as well asciliates (e.g., B. coli). Protozoan can include, but it is not limitedto, Entamoeba coli, Entamoeabe histolitica, Iodoamoeba buetschlii,Chilomastix meslini, Trichomonas vaginalis, Pentatrichomonas homini,Plasmodium vivax, Leishmania braziliensis, Trypanosoma cruzi,Trypanosoma brucei, and Myxoporidia.

The term “algae” as used herein includes, without limitations microalgaeand filamentous algae such as Anacystis nidulans, Scenedesmus sp.,Chlamydomonas sp., Clorella sp., Dunaliella sp., Euglena so., Prymnesiumsp., Porphyridium sp., Synechoccus sp., Botryococcus braunii,Crypthecodinium cohnii, Cylindrotheca sp., Microcvstis sp., Isochrysissp., Monallanthus salina, M. minutum, Nannochloris sp., Nannochloropsissp., Neochloris oleoabundans, Nitzschia sp., Phaeodaciylum tricornutum,Schizochytrium sp., Senedesmus obliquus, and Tetraselmis sueica as wellas algae belonging to any of Spirogyra, Cladophora, Vaucheria,Pithophora and Enteromorpha genera.

The term “fungi” as used herein includes, without limitations, aplurality of organisms such as molds, mildews and rusts and includespecies in the Penicillium, Aspergillus, Acremonium, Cladosporium,Fusarium, Mucor, Nerospora, Rhizopus, Tricophyton, Botryotinia,Phytophthora, Ophiostoma, Magnaporthe, Stachybotrys and Uredinalisgenera.

Discussion:

In accordance with the purpose(s) of the present disclosure, as embodiedand broadly described herein, embodiments of the present disclosure, inone aspect, relate to compositions including a vacancy-engineered(VE)-ZnO nanocomposite, methods of making the composition, methods ofusing the composition, and the like.

In an embodiment, the composition can be used as an antimicrobial agentto kill and/or inhibit the formation of microorganisms on a surface suchas a tree, plant, and the like. An advantage of the present disclosureis that the composition is water soluble, film-forming, hasantimicrobial properties, and is non-phytotoxic. In particular, thecomposition is antimicrobial towards E. coli and X alfalfae and isnonphytotoxic to ornamental vinca sp. In embodiments the composition hasantimicrobial activity towards mircrobial organisms, such as, but notlimited to, Xanthomonas citri subsp. citri, a causal agent of CitrusCanker; Elsinoe fawcetti, a causal agent of citrus scab; and Diaporthecitri, a causal agent of melanose.

In other embodiments, the composition can be used as a systemicantimicrobial agent to kill and/or inhibit the formation and/or growthof microorganisms within a plant, tree, and the like. In suchembodiments, the VE-ZnO nanocomposite particles are able to enter theplant via the roots/vascular system and/or via the leaf stroma. In suchembodiments, the size of the coated VE-ZnO particles are similar to thesize of phloem proteins (e.g., approximately lOnm or less) and can thusbe transported to phloem regions of plant species. This allows theparticles to combat pathogens that reside inside of the plant organism,such as Candidatus Liberibacter asiaticus (CLas), which a causal agentof Huanglongbing (HLB).

In addition, embodiments of the present disclosure provide for acomposition that can be used for multiple purposes. Embodiments of thepresent disclosure are advantageous in that they can substantiallyprevent and/or treat or substantially treat a disease or condition in aplant and act as an antibacterial and/or antifungal, while beingnon-phytotoxic.

In an embodiment, the composition may have an antimicrobialcharacteristic. The phrase “antimicrobial characteristic” can have thefollowing meaning: kills about 70% or more, about 80% or more, about 90%or more, about 95% or more, or about 99% or more, of the microorganisms(e.g., bacteria) on the surface and/or reduces the amount ofmicroorganisms that form or grow on the surface by about 70% or more,about 80% or more, about 90% or more, about 95% or more, or about 99% ormore, as compared to a similar surface without the composition disposedon the surface.

Although not intending to be bound by theory, the unique surface chargeand surface chemistry of the VE-ZnO nanoparticles of the VE-ZnOnanocomposite may be responsible for maintaining good colloidalstability. The high surface area and gel-like structural morphology maybe responsible for the strong adherence properties to a surface, such asa plant surface. The non-phytotoxicity may be attributed to the neutralpH of the VE-ZnO nanocomposite and limited availability of soluble ions.Additional details are described in the Examples.

In an embodiment, the VE-ZnO nanocomposite can include VE-ZnOnanoparticles such as zinc peroxide (ZnO2) or a combination of ZnO andZnO2. In an embodiment, the VE-ZnO nanoparticles have surface defectsassociated with oxygen vacancy, which distinguishes the VE-ZnOnanoparticles from ZnO nanoparticles. UV-Vis studies have shown thatVE-ZnO nanoparticles and ZnO nanoparticles have different opticalcharacteristics, which is indicative of showing that VE-ZnOnanoparticles have surface defects associated with oxygen vacancy.Additional details are provided in the Examples.

In an embodiment, the diameter of the zinc oxide nanoparticles can becontrolled by appropriately adjusting synthesis parameters, such asamounts of the water soluble zinc source, the surface capping agent, andthe oxidizing agent, base, pH, time of reaction, sequence of addition ofthe components, and the like. For example, the diameter of the particlescan be controlled by adjusting the time frame of the reaction. Althoughnot intending to be bound by theory, the superior antimicrobial efficacyof embodiments of the present disclosure can be attributed to thequantum confinement (e.g., size) and surface defect related propertiesof the VE-ZnO nanoparticle. The size of the VE-ZnO nanoparticle mayallow the VE-ZnO nanoparticles to be transported systematically into theplant, reach the phloem tissue, and interact with the pathogen, forexample. In an embodiment, the VE-ZnO nanoparticle can have a diameterof about 1 to 10 nm or about 5 nm or the average diameter is about 5 nm.In embodiments the VE-ZnO nanoparticle can have a diameter of about 10nm or less. In other embodiments, the VE-ZnO nanoparticle can have aplate-like structure, with a thickness of about 10 nm or less, but witha diameter in the sub-micrometer range, e.g., 0.2 to 0.5 micrometers,giving a large surface area.

In an embodiment, the VE-ZnO nanoparticles can be inter-connected to oneanother to form inter-connected VE-ZnO nanoparticle chains. In anembodiment, the VE-ZnO nanocomposite can include a plurality of VE-ZnOnanoparticle chains, where the chains can be independent of one anotheror connect to one or more other chains.

In an embodiment, the VE-ZnO nanoparticles include a coating on thesurface made of the surface capping agent. In an embodiment, the surfacecapping agent includes one or more Zn ion chelating functional groupssuch as carboxyl groups, hydroxyl groups, amines, thiols, and/or acombination of two or more. In an embodiment, the surface capping agentincludes a compound having a carboxyl group and hydroxyl group. In anembodiment the surface capping agent is selected from a small moleculecapping agent such as sodium salicylate, sodium gluconate, as well aspolymers such as chitosan, silica, polyacrylic acid, polyvinyl alcohol,polyacrylamide, polyvinyl pyrrolidine, dextran, polyethelene glycol,dendrimers, and a combination thereof. In an embodiment, the coating cancover the entire surface of the VE-ZnO nanoparticle or a substantialportion (e.g., about 50% or more, about 60% or more, about 70% or more,about 80% or more, about 90% or more, about 95% or more, or about 99% ormore, of the surface of the VE-ZnO nanoparticle) of the surface of theVE-ZnO nanoparticle. In an embodiment, the coating can have a thicknessof about 0.5 nm to 10 nm.

In an embodiment, the VE-ZnO nanocomposite can include the VE-ZnOnanoparticles in a gel-matrix. In an embodiment, the gel matrix caninclude a water soluble zinc source, a surface capping agent, and anoxidizing agent. In an embodiment, the surface capping agent can includecompounds such as those recited above (e.g., sodium salicylate). In anembodiment, the oxidizing agent can be about 10 to 50 or about 25 to 35,weight percent of the VE-ZnO nanocomposite gel matrix.

In an embodiment, the water soluble zinc source can include a watersoluble zinc salt, and organo zinc complexes such as zinc tartarate,zinc citrate, zinc oxalate, zinc acetate, and the like. In anembodiment, the water soluble zinc salt can include zinc nitrate, zincsulfate, and zinc chloride. In an embodiment, the water soluble zincsource can be about 40 to 80 or about 50 to 70, weight percent of theVE-ZnO nanocomposite gel matrix.

In an embodiment, the oxidizing agent is selected from hydrogenperoxide, chlorine, sodium hypochlorite, and a combination thereof. Inan embodiment, the oxidizing agent can be about 10 to 50 or about 25 to35, weight percent of the VE-ZnO nanocomposite gel matrix.

In an embodiment, the method of making a composition can include mixinga water soluble zinc source, a surface capping agent, and an oxidizingagent. In an embodiment, the components are mixed in an aqueous solution(e.g., deionized water). In an embodiment, the components are mixed atroom temperature and after mixing for about 12 to 36 hours, the pH canbe adjusted to about 7.5 with a base such as NaOH. In an embodiment, thecomponents can be simultaneously added together or can be sequentiallyadded together. For example, the surface capping agent and the oxidizingagent can be mixed, and optionally with a base. Then the water solublezinc source can be slowly added dropwise over the course of a fewminutes to an hour, while stirring.

In an embodiment, the oxidizing agent can be about 10 to 50 or about 25to 35, weight percent of the VE-ZnO nanocomposite. In an embodiment, thewater soluble zinc source can be about 40 to 80 or about 50 to 70,weight percent of the VE-ZnO nanocomposite. In an embodiment, theoxidizing agent can be about 10 to 50 or about 25 to 35, weight percentof the VE-ZnO nanocomposite.

In specific embodiments the VE-ZnO nanocomposite includes VE-ZnOparticles having a plate-like structure with a relatively large surfacearea. In embodiments the VE-ZnO particles are made with zinc nitrate,sodium hydroxide and sodium salicylate, resulting in ZnO particles witha coating of sodium salicylate. In some other specific embodiments, theVE-ZnO nanocomposite includes VE-ZnO particles in the 3-8 nm range(average of about 5 nm in diameter) made from zinc nitrate, hydrogenperoxide, sodium hydroxide, resulting in ZnO (and possibly incombination with ZnO2) particles with a coating of sodium salicylate.

Once the components are mixed, the VE-ZnO nanocomposite is formed, wherethe VE-ZnO nanoparticles have a coating formed from the surface cappingagent. The composition can be used as prepared or unbound components(e.g., the water soluble zinc source, the surface capping agent, and theoxidizing agent, and base) can be rinsed off so that only theinter-connected VE-ZnO nanoparticles remain. This process can beperformed using a single reaction vessel or can use multiple reactionvessels. Addition details are provided in the Examples.

In an embodiment, the composition can be disposed on a surface of astructure. In an embodiment, the structure can include plants such astrees, shrubs, grass, agricultural crops, and the like, includes leavesand fruit. In an embodiment, the composition provides uniform plantsurface coverage, substantial uniform plant surface coverage, orsubstantially covers the plant. In an embodiment, the composition can beused to treat a plant having a disease or to prevent the plant fromobtaining a disease.

In an embodiment, the structure can include those that may be exposed tomicroorganisms and/or that microorganisms can grow on, such as, withoutlimitation, fabrics, cooking counters, food processing facilities,kitchen utensils, food packaging, swimming pools, metals, drug vials,medical instruments, medical implants, yarns, fibers, gloves, furniture,plastic devices, toys, diapers, leather, tiles, and flooring materials.In an embodiment, the structure can include textile articles, fibers,filters or filtration units (e.g., HEPA for air and water), packagingmaterials (e.g., food, meat, poultry, and the like food packagingmaterials), plastic structures (e.g., made of a polymer or a polymerblend), glass or glass like structures on the surface of the structure,metals, metal alloys, or metal oxides structure, a structure (e.g.,tile, stone, ceramic, marble, granite, or the like), and a combinationthereof.

In an embodiment, after the composition is disposed on the surface, thestructure may have an antimicrobial characteristic that is capable ofkilling a substantial portion of the microorganisms (e.g., bacteria suchas coli, X alfalfae and S. aureus) on the surface of the structureand/or inhibits or substantially inhibits the growth of themicroorganisms on the surface of the structure. The phrase “killing asubstantial portion” includes killing about 70% or more, about 80% ormore, about 90% or more, about 95% or more, or about 99% or more, of themicroorganism (e.g., bacteria) on the surface that the composition isdisposed on, relative to structure that does not have the compositiondisposed thereon. The phrase “substantially inhibits the growth”includes reducing the growth of the microorganism (e.g., bacteria) byabout 70% or more, about 80% or more, about 90% or more, about 95% ormore, or about 99% or more, of the microorganisms on the surface thatthe composition is disposed on, relative to a structure that does nothave the composition disposed thereon.

In other embodiments, the composition is disposed on the soil or othergrowth substrate in which a plant is growing. In this manner,application facilitates update of the composition by the plant rootsystem and systemic delivery of the composition to various internalregions of the plant. In embodiments, the composition can also be takenup systemically even when delivered to the surface of the plant asdescribed above (e.g., where the plant leaf stomata can take in theparticles of the composition). When delivered systemically, thecomposition may have an antimicrobial characteristic that is capable ofkilling a substantial portion of the microorganisms (e.g., bacteria suchas X cari, E. fawcetti, and D. citri) in the plant systems and/orinhibits or substantially inhibits the growth of the microorganismswithin the plant organism. The phrase “killing a substantial portion”includes killing about 70% or more, about 80% or more, about 90% ormore, about 95% or more, or about 99% or more, of the microorganism(e.g., bacteria) within the plant to which the composition isapplied/delivered to, relative a plant that did not receivedelivery/application of the composition. The phrase “substantiallyinhibits the growth” includes reducing the growth of the microorganism(e.g., bacteria) by about 70% or more, about 80% or more, about 90% ormore, about 95% or more, or about 99% or more, of the microorganismwithin the plant organism.

As mentioned above, embodiments of the present disclosure are effectivefor the treatment of diseases affecting plants such as citrus plants andtrees. In an embodiment, the composition can function as anantibacterial and/or antifungal, specifically, treating, substantiallytreating, preventing or substantially preventing, plant diseases such ascitrus greening (HLB) and citrus canker diseases. The hydroxyl freeradicals, zinc ions, and a combination thereof can act as anantibacterial and/or antifungal for a period of time (e.g., fromapplication to days to months). The design of the compositionfacilitates uniform plant surface coverage or substantially uniformplant surface coverage, and in some embodiments facilitates systemicuptake of the composition by the plant vascular system (e.g., viastromata or root system) and transported to phloem regions of a plant.In an embodiment, the composition that is applied to plants can have asuperior adherence property in various types of exposure to atmosphericconditions such as rain, wind, snow, and sunlight, such that it is notsubstantially removed over the time frame for use of the composition. Inan embodiment, the composition has a reduced phytotoxic effect or isnon-phytotoxic to plants.

Embodiments of the present disclosure can applied on the time framesconsistent with the effectiveness of the composition, and these timeframes can include from the first day of application to about a week,about a month, about two months, about three months, about four months,about five months, about six months, about seven month, or about eightmonths.

In the examples that follow and within the context of use of theforegoing Zinkicide materials the embodiments focus on a Zinkicide SG6material and a Zinciside SG4 material. The difference between theZinkicide SG6 and Zinkicide SG4 composition is that Zinkicide SG6contains hydrogen peroxide but Zinkicide SG4 does not. Zinkicide SG4 ismade of ZnO inorganic crystals (2D plate-like structure, see the HRTEMand SEM images; HRTEM image shows each plate is made of inter-connectingultra-small crystals). When synthesis is carried out in presence ofhydrogen peroxide, this 2D structure is further oxidized to formZnO/ZnO2 (Zn oxide/Zn peroxide) composite material (which appears asparticulate structures in SEM). ZnO is a good stabilizer for hydrogenperoxide. ZnO2 is a fairly stable inorganic compound. ZnO and ZnO2havedifferent crystal structures which produces surface defects in thecomposite. ZnO can produce ROS (such as hydrogen peroxide) with somesurface defects. However, the ROS production is drastically enhanced inZnO/ZnO2 composite as it has more surface defects and in addition thecomposite contains peroxide. ZnO2 decomposes to ZnO over time and thisprocess is dependent on the environmental conditions.

EXAMPLE 1

Materials and Methods

Formulation abbreviations: Z-SG-1, ZPER-SG-1, ZPER-SG-2, ZSAL-SG-2,ZPSAL-SG-3, ZPSAL-SG-4, ZPSAL-SG-5, ZPSAL-SG-6, ZPSAL-SG-7

Detailed nanoformulation synthesis procedure: Z-SG-1, ZPER-SG-1,ZSAL-SG-2, ZPSAL-SG-3 and ZPSAL-SG-4 synthesis procedure:

In a glass beaker, take 50 ml deionized water, 5 ml Zn nitrate stocksolution (59 weight %), add 1M NaOH dropwise under magnetic stirringuntil pH is 7.5. Then divide into 5 equal parts:

-   -   Z-SG-1: no treatment    -   ZPER-SG-1: add 2 ml hydrogen peroxide (30%)    -   ZSAL-SG-2: add 1 ml of sodium salicylate solution (32.8 weight        %)    -   ZPSAL-SG-3: add 1 ml of sodium salicylate solution (32.8 weight        %), wash to remove unbound sodium salicylate solution, add 2 ml        hydrogen peroxide (30%)

ZPSAL-SG-4: add 2 ml hydrogen peroxide (30%), stir for 2 hours, wash toremove unbound hydrogen peroxide, add 1 ml of sodium salicylate solution(32.8 weight %), wash

ZPER-SG-2 and ZPSAL-SG-5 Synthesis Procedure:

In a glass beaker, take 40 ml deionized water, 10m1 hydrogen peroxide(30%) and 5 ml Zn nitrate stock solution (59 weight %). Adjust pH to 7.5with 1N NaOH. Then, divide into 2 equal parts

-   -   ZPER-SG-2: no treatment    -   ZPSAL-SG-5: add 2.5 ml sodium salicylate solution (32.8 weight        %), check pH-adjust to 7, let stir overnight.

ZPSAL-SG-6 Synthesis Procedure (Coated VE-ZnO)**:

In a glass beaker, take 40 ml deionized water, 10 ml hydrogen peroxide(30%), 2.5 ml sodium salicylate solution (32.8 weight %) and 5 ml ZnNitrate stock solution (59 weight %). Magnetically stir overnight thenadjust pH to 7.5 with 1N NaOH (approximately 25 ml).

**Coated ZnO material is identical to coated VE-ZnO except that itcontains no hydrogen peroxide.

ZPSAL-SG-7 synthesis procedure: In a glass beaker, take 40 ml deionizedwater, 10 ml hydrogen peroxide (30%), 2.5 ml sodium salicylate solution(32.8 weight %) and add approximately 20 ml 1N NaOH. Then add dropwise(very carefully and slowly; a few drops per minute) Zn Nitrate solution(59 weight %) under vigorous magnetic stirring until pH is reached to7.5.

FIG. 1 illustrates: (a) A representative HRTEM image of Zinkicide™ SG4showing plate-like faceted structure in the sub-micron size range. (b)High-magnification image of coated ZnO material shows appearance of bothpolycrystalline and amorphous regions within a plate structure. FieldEmission Scanning Electron Microscopy (FE-SEM) image of the material areshown in image (c) and (d).

FIG. 2 illustrates: (a) A representative low-magnification HRTEM imageof Zinkicide™ SG6 material showing gel-like network of inter-connectingultra-small size (<5 nm) crystalline sol particle clusters. (b)High-magnification image of Zinkicide™ SG6 material. Inset showscrystalline lattice fringe of one of Zinkicide™ SG6 sol particles. Note:one nm is a billionth of a meter. Field Emission Scanning ElectronMicroscopy (FE-SEM) images of the material are shown in image (c) and(d).

FIG. 3 illustrates: (a) A representative HRTEM image of Nordox 30/30 WGmaterial showing polydispersed structure in the size ranging from nanoto micron size. (b) High-magnification image of Nordox material showsappearance of highly crystalline structure (see inset; HRTEM-SAEDpattern showing bright spots confirming crystallinity). Field EmissionScanning Electron Microscopy (FE-SEM) images of the material are shownin image (c) and (d).

FIGS. 4A through 4E illustrate phytotoxicity results of variouscoatings. In particular, FIG. 4 illustrates a phytotoxicity assessmentof: (a) uncoated (b) surface coated ZnO, (c) surface coated VE-ZnO, (d)Nordox, and (e) Kocide 3000 materials. Formulations were applied atspray rate of 790 ppm metallic Zn. Digital photographs showing no planttissue damage (—) occurred even after 72 hours.

FIG. 5 illustrates the growth inhibition with Alamar blue Assay of E.coli against VE-ZnO, coated ZnO, Nordox, and Kocide 3000.

FIG. 6 illustrates E. coli growth curves in presence of Zinkicide™ of E.coli against VE-ZnO, coated ZnO, Nordox, and Kocide 3000.

FIG. 7 illustrates E. coli viability in presence of Zinkicide™materials. In particular, FIG. 7 illustrates viability of E. coliagainst VE-ZnO, coated ZnO, Nordox and Kocide 3000.

FIG. 8 illustrates direct evidence of ROS generation by the coatedVE-ZnO material. FIG. 8 illustrates transmission spectra ofmixed-valence ceria and ceria treated with surface coated VE-ZnOmaterial. Ceria and VE-ZnO are whitish in color. However, when combinedtogether an intense red color develops. A clear shift of ceriatransmission wavelength towards longer wavelength was observed,confirming conversion of Ce³⁺ to Ce⁴⁺ state upon reaction with ROS(produced by the surface coated VE-ZnO material).

FIGS. 9A and 9B illustrate HRTEM-EDX spectra of surface coated VE-ZnOand ZnO. FIG. 9 illustrates a representative HRTEM-EDX spectra ofsurface coated A VE-ZnO and B surface coated ZnO materials.Characteristic elemental peaks of Zn and oxygen were found in thespectra. Au peak is originated from the HRTEM Au grid substrate.

FIGS. 10A and 10B illustrate x-ray photoelectron spectroscopy (XPS)results of surface coated VE-ZnO and ZnO. In particular, FIG. 10illustrates XPS results of surface coated: (a) VE-ZnO and (b) surfacecoated ZnO materials. Characteristic peak of Zn (II) oxidation state wasobserved.

FIG. 11 illustrates a schematic representation of VE ZnO (“Zinkicide”)nanoparticle composite (nanocomposite).

FIG. 12 illustrates Zinkicide™ leaf washoff properties.

FIG. 13 illustrates Zinkicide™ properties relative to snow pea seedgermination.

FIG. 14A, FIG. 14B and FIG. 14C illustrate experimental methodology andresults of measuring uptake of Zinkicide™ into tomato plants.

FIG. 15 shows UV-visible optical spectra characteristics of a Zinkicide™material in accordance with the embodiments.

FIG. 16A and 16B shows a fluorescence emission spectrum of Zinkicidematerials in accordance with the embodiments.

FIG. 17A and 17B illustrate FT-IR spectra of surface coated ZnO, surfacecoated VE-ZnO and the surface coating agent. FTIR results show that thecoating agent is present in both ZnO and VE-ZnO materials. In FIG. 17A,the curve that corresponds with the peak at 1600 cm-1 corresponds withthe surface coating agent. The curve that corresponds with the peak at1350 cm-1 corresponds with surface coated ZnO and the remaining curvewhich does not include a deep peak corresponds with surface coatedVE-ZnO. In FIG. 17B, the curve that corresponds with the peaek at3500cm-1 cororesponds with the surface coated VE-ZnO, the curve thatcorresponds with thet peak at 2000 corrresponds with surface coatingagent and the remaining curve corresponds with the surface coatedVE-ZnO.

FIG. 18 illustrates an XRD of surface coated VE-ZnO. XRD patternrevealing 200 (strong), 220 and 311 reflection peaks VE-ZnO at 2 ⊖ valueof ˜36°, 54° and 64° were observed. These peaks are characteristic toZnO material with oxygen vacancy. The appearance of XRD peak at 20 valueof ˜17° has not been assigned yet (possibly originating from the surfacecoating agent).

FIG. 19A and 19B illustrate Raman spectra of (a) surface coated VE-ZnOand (b) surface coated ZnO materials. Appearance of strong ˜840 cm⁻¹Raman peak is characteristic to VE-ZnO O—O stretching vibration ofperoxide (an active ROS). No such peak is present in surface coated ZnOmaterial.

FIG. 20A and 20B illustrate DLS particle size distribution of (a)surface coated ZnO and (b) surface coated VE-ZnO materials. Narrowparticle size distribution of VE-ZnO material is indicative of smallerand uniform-size cluster formation

Table 1 illustrates the minimum inhibitory concentration against E. colifor various agents.

TABLE 1 MIC of surface coated VE-ZnO, coated ZnO, surface capping agent,Kocide 3000, and Nordox against E. coli Tested Material MIC (μg/mL) inmetallic Zn or Cu Surface coated ZnO 750 Surface coated VE-ZnO 93.75Capping Agent 3000 Kocide 3000 1000 Nordox 750

EXAMPLE 2

Materials/Methods:

This example describes the testing of various applications andeffectiveness of two formulations of the VE-ZnO nanocomposites of thepresent disclosure. The formulations correspond to the particleformulations described in Example 1 above as follows:

-   -   Zinkicide™ SG4 corresponds to ZSAL-SG-2 in Example 1, above    -   Zinkicide™ SG6 corresponds to ZPSAL-SG-6 in Example 1, above.

More specifically, in the present example, SG4 (3.14 gallon preparation)is prepared as follows (2 hr preparation time):

1. DI water—3.75 L

2. Zinc nitrate hexahydrate solution—1.25 L (59 wt % solution in DIwater)

3. Sodium hydroxide—6.25 L (1M solution)

4. Sodium salicylate—625 mL (32.8 wt % solution in DI water)

In the present example, Zinkicide SG6 (3.14 gallon preparation) isprepared as follows:

1. DI water—1.25 L

2. Hydrogen peroxide (30% solution as supplied; 2.5 L)

3. Sodium salicylate—625 mL (32.8 wt % solution in DI water)

4. Zinc nitrate hexahydrate—1.25 L (59 wt % solution in DI water)

5. Sodium hydroxide—6.25 L (1M solution)

6. pH then further adjusted to 7.5 by adding 115 mL of 5M NaOH solution

7. Although not discussed in detail the ZnO formulations of this Exampleare the VE-ZnO particles described in detail in the application, above.This formulation as well as the size and shape of the particles andother features of the novel VE-ZnO formulations of the presentdisclosure distinguish these formulations from ZnO components of otherproducts, such as the Nordox® 30/30 used as a comparison in thisExample.

Discussion:

In the present example, the SG4 and SG6 formulations both outperformedprior art Nordox formulations that contain copper oxide/zinc oxide incombination. The formulations of the present disclosure do not containcopper, which reduces potential copper soil build up as well as otherproblems such as copper toxicity. In the attached example the SG4 andSG6 applied as a spray to plant surfaces (stems, leaves, fruits, etc.)outperformed the comparison products and control . Additionalexperiments were conducted where SG6 was applied systemically by soildrench (to allow systemic uptake by the plant vascular system). In thesetrials, the SG6 formulation was shown to have systemic uptake andeffect, demonstrating that the VE-ZnO formulations of the presentdisclosure can have systemic as well as surface effectivity, and can beapplied to surfaces (e.g., spray, powder, etc.) or to soil or otherplant growth substrate/medium (e.g., hydroponic or other growthconditions where soil is not used as the growth substrate) to be takenup by plant roots and/or plant vascular system for systemic action.Applied in this manner “drench” application, the SG6 formulationsoutperformed both traditional protective coating formulations (such ascopper, e.g., Nordox®) and other fully or locally systemic formulations(e.g., Firewall™). Thus the Ve-ZnO formulations of the presentdisclosure offer additional benefits in that they can provide protectionand antimicrobial efficacy both as a protective coating application aswell as a systemic protection (either through absorbance through leafstromata or uptake via plant vascular system).

It should be noted that ratios, concentrations, amounts, and othernumerical data may be expressed herein in a range format. It is to beunderstood that such a range format is used for convenience and brevity,and thus, should be interpreted in a flexible manner to include not onlythe numerical values explicitly recited as the limits of the range, butalso to include all the individual numerical values or sub-rangesencompassed within that range as if each numerical value and sub-rangeis explicitly recited. To illustrate, a concentration range of “about0.1% to about 5%” should be interpreted to include not only theexplicitly recited concentration of about 0.1 wt % to about 5 wt %, butalso include individual concentrations (e.g., 1%, 2%, 3%, and 4%) andthe sub-ranges (e.g., 0.5%, 1.1%, 2.2%, 3.3%, and 4.4%) within theindicated range. In an embodiment, the term “about” can includetraditional rounding according to measurement techniques and thenumerical value. In addition, the phrase “about ‘x’ to ‘y’” includes“about ‘x’ to about ‘y’”.

Many variations and modifications may be made to the above-describedembodiments. All such modifications and variations are intended to beincluded herein within the scope of this disclosure and protected by thefollowing claims.

EXAMPLE 3

Rainfastness of Zinkicide

-   -   Sour Orange root stock plants (N=3)    -   Materials tested were Zinkicide SG6-S, G and U versions—Zn        Nitrate based    -   Applications were made using a pressurized sprayed bottle (Home        Depot) at 800 ppm metallic Zn (similar to application rate in        Citrus Canker Trial) until plants were fully covered and        dripping.    -   After spraying, plants were allowed to air dry for 24hrs before        starting simulated rainfall.    -   Used 80 gallon/hr fountain pump to stimulate rainfall from a PVC        tube with holes.    -   Dispensed ˜4 gallons of water during each rainfall for each        group of plants.    -   Rainfalls were 24 hrs apart to allow plants to dry.    -   After final rainfall and allowing drying, ˜2.0 g of leaves were        removed from different heights and angles of the plant.    -   Leaves were placed in a 50 mL conical tube and rotated at 15 rpm        for 1 hr with 30 mL of 1% HCL.    -   After rotation, solution was filtered using Whatman filter paper        and filtrate was analyzed for Zn with Atomic Absorption        Spectroscopy (AAS).    -   Untreated controls were analyzed and showed Zn concentration        below the detection limit (0.8 ppm).

Results are shown in FIG. 12 which illustrates substantial Zinkicidewash off.

EXAMPLE 4

Seed Germination and Seedling Growth

-   -   Germination test monitored over 5 days    -   Concentration used: 50, 100, 250 and 500 ppm metallic Zn    -   Materials tested:        -   Zinkicide SG-6 (Original)        -   Zinkicide SG-4 (Zinkicide with no oxidizing agent)        -   Zinkicide SG-6 (No capping agent)        -   Zinkicide SG-4 (No capping agent)        -   Zinc Peroxide (Sigma-Aldrich)        -   Urea Hydrogen Peroxide Mixture

Results of Example 4 seed germination and seedling growth are foundwithin the chart of FIG. 13. In turn the chart of FIG. 13 shows ingeneral that a germination percentage of a snow pea seed may bedecreased when treating the snow pea seed with a Zinkicide material.

EXAMPLE 5

Uptake of Zinkicide in tomato plants

FIG. 14A, FIG. 14B and FIG. 14C show experimental methodology andresults of measuring uptake of Zinkicide into tomato plants.

Therefore, at least the following is claimed:
 1. A compositioncomprising a vacancy-engineered (VE)-ZnO nanocomposite including aplurality of interconnected VE-ZnO nanoparticles, wherein the pluralityof interconnected VE-ZnO nanoparticles has a plurality of surfacedefects associated with an oxygen vacancy, wherein at least one of: theVE-ZnO nanoparticles each have a diameter of other than about 3 to 8 nm;and the VE-ZnO nanoparticles each do not include a coating of a surfacecapping agent having one or more Zn ion chelating functional groups. 2.The composition of claim 1 wherein the surface capping agent is selectedfrom the group consisting of sodium salicylate, sodium gluconate,chitosan, silica, polyacrylic acid, polyvinyl alcohol, polyacrylamide,polyvinyl pyrrolidine, dextran, polyethelene glycol, dendrimer, and acombination thereof
 3. The composition of claim 1 wherein, if present:the VE-ZnO nanoparticles have an average diameter of about 5 nm; and thecoating covers the surface of each of the VE-ZnO nanoparticles.
 4. Thecomposition of claim 1 wherein the coating has a thickness of about 0.5nm to 10 nm.
 5. The composition of claim 1 wherein the VE-ZnOnanocomposite is disposed in a gel matrix including hydrogen peroxide.6. The composition of claim 5 wherein hydrogen peroxide is about 10 to50 weight percent of the VE-ZnO nanocomposite.
 7. The composition ofclaim 1 wherein the VE-ZnO nanocomposite is disposed in a gel matrixincluding hydrogen peroxide and sodium hydroxide.
 8. The composition ofclaim 7 wherein hydrogen peroxide is about 10 to 50 weight percent ofthe VE-ZnO nanocomposite and wherein sodium hydroxide is about 10 to 50weight percent of the VE-ZnO nanocomposite.
 9. The composition of claim1 wherein the composition has antimicrobial characteristics towards oneor more species of microbial organism selected from the group consistingof: E. coli, X alfalfae, S. aureus, X citri, E. fawcetti, CandidatusLiberibacter asiaticus, and D. citri.
 10. The composition of claim 1wherein the composition is non-phytotoxic to ornamental vinca sp, ‘RayRuby’ grapefruit, ‘Pineapple’ sweet orange.
 11. A method, comprising:applying a composition to a plant, wherein the composition has avacancy-engineered (VE)-ZnO nanocomposite including a plurality ofinterconnected VE-ZnO nanoparticles, wherein the plurality ofinterconnected VE-ZnO nanoparticles has a plurality of surface defectsassociated with an oxygen vacancy, wherein at least one of: theplurality of VE-ZnO nanoparticles does not include a coating of asurface capping agent having one or more Zn ion chelating functionalgroups; and the plurality of VE-ZnO nanoparticles does not include asize range of about 3 to about 8 nanometers; and killing a substantialportion of a microorganism or inhibiting or substantially inhibiting thegrowth of the microorganisms on the surface or within the plant.
 12. Themethod of claim 11 wherein the microorganism is a bacterium.
 13. Themethod of claim 11, wherein the microorganism selected from the groupconsisting of E. coli, B. subtilis, Xanthomonas sp, CandidatusLiberibacter spp, and S. aureus.
 14. The method of claim 11 whereinapplying includes application of the composition to the growth substratein which a plant is growing.
 15. The method of claim 14 wherein thegrowth substrate is soil and delivery includes applying the compositionto the soil surrounding the plant.
 16. The method of claim 11 whereinapplying includes forming a film of the composition on the surfaces ofthe plant.
 17. The method of claim 11, wherein applying includes forminga substantially uniform plant surface coverage.
 18. The method of claim11 wherein the VE-ZnO nanoparticle has a diameter of about 1 to 10 nm.19. The method of claim 11 wherein the VE-ZnO nanoparticle has aplate-like structure.
 20. A method of making a composition, comprising:mixing in an aqueous solution a water soluble zinc source and anoxidizing agent; and forming in the aqueous solution avacancy-engineered (VE)-ZnO nanocomposite including a plurality ofinterconnected VE-ZnO nanoparticles, wherein each of the plurality ofVE-ZnO nanoparticles has a plurality of surface defects associated withan oxygen vacancy, wherein at least one of: the mixing does not includea surface capping agent that has both a carbonyl group and a hydroxylgroup; and the forming provides the plurality of VE-ZnO nanoparticlesthat each has a diameter of other than about 1 to 10 nm.
 21. The methodof claim 20 wherein the oxidizing agent is about 10 to 50 weight percentof the V5E-ZnO nanocomposite.
 22. The method of claim 20 wherein theoxidizing agent is selected from the group consisting of: hydrogenperoxide, chlorine, sodium hypochlorite and a combination thereof, andwherein the surface capping agent is selected from the group consistingof sodium salicylate, sodium gluconate, chitosan, silica, polyacrylicacid, polyvinyl alcohol, polyacrylamide, polyvinyl pyrrolidine, dextran,polyethelene glycol, dendrimers, and a combination thereof.
 23. A methodof making a composition comprising: mixing a water soluble zinc sourceand an oxidizing agent selected from hydrogen peroxide, sodiumhypochlorite, or both; and forming a vacancy-engineered (VE)-ZnOnanocomposite including interconnected VE-ZnO nanoparticles, wherein theVE-ZnO nanoparticles have surface defects associated with oxygenvacancy.
 24. The method of claim 23, wherein the VE-ZnO nanoparticleshave a plate-like structure.
 25. The method of claim 23, wherein theVE-ZnO nanoparticles have a diameter of about 1 nm to about 10 nm.
 26. Amethod for applying a treatment material to a plant comprising injectinga part of the plant with a fluid composition comprising the treatmentmaterial.
 27. The method of claim 26 wherein the treatment materialcomprises a Zinkicide material.
 28. The method of claim 26 wherein thepart of the plant is a stem.